Project Summary The blockade of immune-checkpoint pathways has emerged as a promising therapeutic strategy for a variety of cancers. However, the diverse tumor responses to immunotherapy seen in preclinical and clinical studies prompt the development of combination immunotherapies that can be tailored to the complex immune milieu of individual patients. On the other hand, the severe adverse effects associated with the combination therapies with multiple antagonist antibodies address the necessity for alternative safe and effective therapeutic approaches. In light of this, we aim to develop a hybrid nanoparticle-viral vector system for CRISPR/Cas9- based in vivo therapeutic genome editing, which will be used for multiplexed disruption of immune suppressive pathways in the tumor microenvironment. CRISPR/Cas9 systems are very efficient in generating DNA double- strand breaks, thus disrupting genes through the non-homologous end-joining (NHEJ) pathway. However, inducing uncontrolled CRISPR/Cas9 activities in vivo may lead to systemic genotoxicity. We will develop a novel in vivo gene delivery system that integrates a baculoviral vector (BV) with magnetic nanoparticles (MNPs). Our studies have shown that by taking advantage of the interplay between the MNP-mediated BV margination and endocytosis and the innate immunity against insect viruses, this delivery system can provide spatial and temporal control of CRISPR/Cas9 activity. We will use the MNP-BV system to deliver optimized CRISPR/Cas9 for gene disruption of immune suppressive signals PD-L1 and TGF-? in the tumor tissue. We will evaluate CRISPR/Cas9 induced anti-tumor immune responses using two well-established mouse models, an immunogenic model (MC38) where monotherapy with PD-1 blockade is sufficient, and non-immunogenic models pancreatic ductal adenocarcinoma (PDAC) which portrays most non-immunogenic human solid tumors that require combination strategies. In aim 1 studies, we will design and optimize CRISPR/Cas9 gRNAs for targeting PD-L1 and TGF-?, package Cas9 and gRNAs into a BV vector, and construct the MNP- BV system. In aim 2 studies, we will evaluate MNP-BV-induced multiplexed gene disruption in cell culture, and determine the on-target and off-target indel rates. In aim 3 studies, we will test the controlled in vivo delivery of CRISPR/Cas9, determine the immunological and therapeutic effects of local gene disruption vs. systemic blockade with antagonist antibody in mouse tumor models. The success of the proposed studies will provide a multiplexed intratumoral immunoengineering platform and pave the way for the clinical translation of a highly efficient in vivo genome editing strategy for personalized cancer immunotherapy.